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Laser tech to detect pathogens without damaging colonies

By Joe Whitworth+

10-Feb-2014
Last updated on 10-Feb-2014 at 12:21 GMT

A laser optical sensor has been created to screen pathogen colonies on an agar plate without damaging the colonies.

Bacterial rapid detection using optical scatter technology (BARDOT) is suitable for real-time on-plate detection (screening) of Salmonella colonies (88 to 100% accuracy) from classical microbiological culture-based methods.

BARDOT is a label-free nondestructive method in which a 635-nm laser beam passes through the center of a colony and generates a unique scatter signature (fingerprint) that can be used for detection and identification employing a scatter signature classification library.

Serovar differentiation

The objective was to investigate BARDOT’s abilities to differentiate the top 20 S. enterica subsp. Enterica serovars, which are responsible for 72.8% of Salmonella isolates reported to Centers for Disease Control and Prevention (CDC), from bacterial species and to detect and identify them.

Researchers found the similarities between the optical phenotypes of strains analyzed by BARDOT were in general agreement with genotypes analyzed by pulsed-field gel electrophoresis (PFGE).

The ability of BARDOT to detect Salmonellain spinach, chicken breast with a high natural microbial background level, and in a low-moisture model food system (peanut butter) was examined.

It detected Salmonella in inoculated peanut butter, spinach, and raw chicken within 24 hours, and the results were in agreement with the standard USDA FSIS method completed in about 72 hours.

The data shows that the system can accurately detect most of the Salmonella serovars tested in the 20 most prevalent serovars (positive predictive value [PPV] or classification precision level reaching 86%) and all serovars in the top eight group (PPVs ranging from 68 to 93%).

Testing the method

Researchers tested scatter signatures formed by colonies of six randomly picked Salmonella serovars grown on brain heart infusion, brilliant green, xylose lysine deoxycholate, and xylose lysine tergitol 4 (XLT4) agar plates.

Highly accurate discrimination (95.9%) was possible by using scatter signatures collected from the colonies grown on XLT4.

The technique has been demonstrated before to differentiate Escherichia, Listeria, Salmonella, Staphylococcus, and Vibrio at the genus level with 90 to 99% accuracy.

It was successful in differentiating Listeria species and detected Listeria monocytogenes on agar plates containing bacteria captured and preconcentrated with paramagnetic beads.

However, BARDOT’s ability to identify bacteria at the serovar level had not been fully explored.

Salmonella detection and identification involves culture-based methods coupled with metabolic fingerprinting, immunoassays, and nucleic acid-based methods.

Other biosensor-based methods based on fiber-optic, surface plasmon resonance, impedance, magnetoelastic biosensors and nanoparticle-based DNA sensors have been proposed but these methods can detect only a selected serovar and do not allow pathogen recovery.

A new generation of BARDOT is being manufactured by Advanced Bioimaging Systems, Indiana with an attached incubator that holds petri plates for high throughput automated screening of plates and minimal user intervention.

Source: American Society for Microbiology

Online, ahead of print DOI:10.1128/mBio.01019-13

Laser Optical Sensor, a Label-Free On-Plate Salmonella enterica Colony Detection Tool”

Authors: Atul K. Singh, Amanda M. Bettasso, Euiwon Bae, Bartek Rajwa, Murat M. Dundar, Mark D. Forster, Lixia Liu, Brent Barrett, Judith Lovchik, J. Paul Robinson, E. Daniel Hirleman, Arun K. Bhuniaa

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